We have continued our investigations of the determinants of G-protein-coupled receptor (GPCR) selectivity for G-protein using recombinant expressed GPCRs and G-protein alpha subunit chimeras to map the contact site(s) on the alpha subunit responsible for receptor-selective interactions. Previously, we had constructed chimeras between G-alpha-i1 and G-alpha-q containing differing lengths of G-alpha-q sequence in place of the C-terminal sequence of G-alpha-i1 and testing for capacity to be activated by the human 5HT1A and 5HT2c receptors which are uniquely selective for the respective G-alpha-i1 and -q subunit types. Those experiments mapped the selective contact site(s) to include at least 50 residues of the carboxyl terminus of G-alpha-q. By co-expression of the chimeric G-alpha constructs with the yeast N-myristoyltransferase enzyme in e.coli hosts and purifying the myristoylated (myr-G-alpha) proteins to homogeneity, we have now refined this mapping. Our current constructs include chimeric G-alpha subunits containing G-alpha-q sequence internal to the C-terminus. This has defined the selective contact surface to be two beta-stranded stretches of the G-alpha subunit termed beta5 and beta6 and it has excluded the carboxyl terminal 25 amino acid residues as providing selective receptor contact(s). [unreadable] We have also examined the role of the lipid modification of the G-alpha-i1/q chimeric subunit chains in receptor interaction by separating the non-myr- and myr-G-alpha products. In each case, only the myr-alpha was capable of receptor activation, either by 5HT1A receptors or cephalopod rhodopsin for the wild-type G-alpha-i1 or 5HT2c receptors and cephalopod rhodopsin for the G-alpha-i1/q chimeric constructs. Further, chimeric constructs of G-alpha-s containing G-alpha-i1 sequence replacing the N-terminal 37 residues expressed in e.coli allowed us to perform a similar experiment testing beta2-adrenergic receptor activation of unmodified and lipid-modified G-alpha-s. As for the G-alphai1/q chimeras, only the myr-G-alphai1/s chimeras were activated by the beta2-adrenergic receptors. These experiments also determined that the contact(s) between the G-alpha-s constructs and beta2-adrenergic receptor involved at least three distinct sites on the G-alpha chain, since rotations of the amino terminal G-alphai1 alpha helix (producing stereo-enantiomers) disrupted activation by the beta2-adrenergic receptor while preserving the capacity of the G-alpha chains to bind GTP and interact with the G-beta-gamma dimer (manuscript in preparation).[unreadable] As a part of the examination of receptor-selective G-protein interaction, we have collaborated with the laboratory of Reinhard Grisshammer who has produced a homogeneous, purified preparation of neurotensin receptor (NTR) subtype NTS1 suitable for crystallization. We have further examined its suitability for the formation of stable complexes with G-protein subunits. Our initial results have identified detergent conditions that allow the productive, ligand regulated interaction of G-alpha-q and G-beta-gamma with NT-R. Under optimal conditions, NT stimulates the NT-R catalyzed rate exchange of GTP for GDP by 50-fold, with Km for G-alpha-q of about 300 nM and G-beta1-gamma2 of about 200 nM. Thus, it is feasible to prepare the complex of NT-R with G-alpha-q and/or G-beta1-gamma2 in defined detergent conditions. The biophysical characterization of these complexes under these conditions provided the surprising result that the NT-R is a dimeric complex. In the absence of G-protein, this dimer displays positive cooperative binding for NT. The apparent Kd for the monomer-dimer interaction is in the low nM range, which is 2 orders of magnitude higher affinity than any found so far for transmembrane proteins in detergent solutions. Even more surprising, while the NT-R clearly prefers a dimer organization, the monomer displays both a higher affinity and considerably higher catalytic activity towards G-alpha-q than does the monomer. Our current studies are aimed at the definition of the stoichiometry of G-protein subunit interaction with the dimeric NT-R structure and an elucidation of the molecular mechanism for G-protein activation.[unreadable] [unreadable] Lastly, in collaboration with Dr. Susan Sullivan, NIDCD, we have examined the signaling properties of novel constructs produced from the human sweet and amino acid sensing T1Rs (T1R1-3). These GPCRs belong to the family3, which is characterized by an autonomously folded amino-terminal domain which is the site(s) for ligand regulation. The T1R taste receptors are heterodimeric complexes of T1R1/T1R3 or T1R2/T1R3. Thus, they present many unique challenges for expression and molecular characterization. Therefore, we have constructed truncated receptor structures from these which consist of the transmembrane helix bundle (7TM) domain, the portion which is responsible for G-protein interaction, with which to test the T1R-G-protein selectivity. We were unable to select stable mammalian cell clones expressing these constructs. Even transient expression in mammalian cell hosts lead to a lack of viability for the T1R1 and T1R2 7TM domains. Therefore, we constructed baculovirus vectors for their expression in insect cells. Using membranes derived from Sf9 cells expressing the T1R 7TM domains, we find that both T1R1 and T1R2 constructs constitutively activate G-proteins of the alphai family (alpha-i1, alpha-o, alpha-gustducin and alplha-transducin). None of the identified sweet tastants or allosteric regulators modulate the activation for these receptors. These data suggest that the extracellular domains for the T1Rs (and likely the entire family3) are inhibitory constraints on the 7TM domain. Further, these experiments define a G-protein pathway which is likely to be mediated by the common beta-gamma component of the alphai family. This result is entirely consistent with our examination of the signaling properties of the human T2R bitter taste receptors, for which we have succeeded in identifying ligands capable of activating G-alpha subunits in in vitro screening for the T2R14 (aristolochic acid), T2R43 (aristolochic acid), T2R47 (denatonium) and T2R7 (strychnine, choroquine). The human T2R receptors also activate the entire G-alpha-i family of G-proteins, suggesting the sweet and bitter taste transduction mechanisms are shared, and that the sensory modalities are encoded by which type of sensory cell is stimulated.